Estrogen contributes to sex differences in M2a macrophages during multi-walled carbon nanotube-induced respiratory inflammation.

Lung diseases characterized by type 2 inflammation are reported to occur with a female bias in prevalence/severity in both humans and mice. This includes previous work examining multi-walled carbon nanotube (MWCNT)-induced eosinophilic inflammation, in which a more exaggerated M2a phenotype was observed in female alveolar macrophages (AMs) compared to males. The mechanisms responsible for this sex difference in AM phenotype are still unclear, but estrogen receptor (ER) signaling is a likely contributor. Accordingly, male AMs downregulated ERα expression after MWCNT exposure while female AMs did not. Thus, ER antagonist Fulvestrant was administered prior to MWCNT instillation. In females, Fulvestrant significantly attenuated MWCNT-induced M2a gene expression and eosinophilia without affecting IL-33. In males, Fulvestrant did not affect eosinophil recruitment but reduced IL-33 and M2a genes compared to controls. Regulation of cholesterol efflux and oxysterol synthesis is a potential mechanism through which estrogen promotes the M2a phenotype. Levels of oxysterols 25-OHC and 7α,25-OHC were higher in the airways of MWCNT-exposed males compared to MWCNT-females, which corresponds with the lower IL-1ß production and greater macrophage recruitment previously observed in males. Sex-based changes in cholesterol efflux transporters Abca1 and Abcg1 were also observed after MWCNT exposure with or without Fulvestrant. In vitro culture with estrogen decreased cellular cholesterol and increased the M2a response in female AMs, but did not affect cholesterol content in male AMs and reduced M2a polarization. These results reveal the modulation of (oxy)sterols as a potential mechanism through which estrogen signaling may regulate AM phenotype resulting in sex differences in downstream respiratory inflammation.

Nanoparticle-Induced Airway Eosinophilia Is Independent of ILC2 Signaling but Associated With Sex Differences in Macrophage Phenotype Development.

The majority of lung diseases occur with a sex bias in terms of prevalence and/or severity. Previous studies demonstrated that, compared with males, female mice develop greater eosinophilic inflammation in the airways after multiwalled carbon nanotube (MWCNT) exposure. However, the mechanism by which this sex bias occurs is unknown. Two immune cells that could account for the sex bias are type II innate lymphoid cells (ILC2s) and alveolar macrophages (AMs). In order to determine which immune cell type was responsible for MWCNT-induced airway eosinophil recruitment and subsequent sex differences in inflammation and disease, male and female C57BL/6 mice were exposed to MWCNTs (2 mg/kg) via oropharyngeal aspiration, and the respiratory immune response was assessed 7 d later. Greater eosinophilia and eotaxin 2 levels were observed in MWCNT-treated females and corresponded with greater changes in airway hyperresponsiveness than those in MWCNT-treated males. In MWCNT-treated females, there was a significant increase in the frequency of ILC2s within the lungs compared with control animals. However, depletion of ILC2s via α-CD90.2 administration did not decrease eosinophil recruitment 24 h and 7 d after MWCNT exposure. AMs isolated from control and MWCNT-treated animals demonstrated that M2a macrophage phenotype gene expression, ex vivo cytokine production, and activation of (p)STAT6 were upregulated to a significantly greater degree in MWCNT-treated females than in males. Our findings suggest that sex differences in AM phenotype development, not ILC2 signaling, are responsible for the observed female bias in eosinophilic inflammation after MWCNT inhalation.

Lysosomal BK channels facilitate silica-induced inflammation in macrophages.

BACKGROUND: Lysosomal ion channels are proposed therapeutic targets for a number of diseases, including those driven by NLRP3 inflammasome-mediated inflammation. Here, the specific role of the lysosomal big conductance Ca2+-activated K+ (BK) channel was evaluated in a silica model of inflammation in murine macrophages. A specific-inhibitor of BK channel function, paxilline (PAX), and activators NS11021 and NS1619 were utilized to evaluate the role of lysosomal BK channel activity in silica-induced lysosomal membrane permeabilization (LMP) and NLRP3 inflammasome activation resulting in IL-1ß release. METHODS: Murine macrophages were exposed in vitro to crystalline silica following pretreatment with BK channel inhibitors or activators and LMP, cell death, and IL-1ß release were assessed. In addition, the effect of PAX treatment on silica-induced cytosolic K+ decrease was measured. Finally, the effects of BK channel modifiers on lysosomal pH, proteolytic activity, and cholesterol transport were also evaluated. RESULTS: PAX pretreatment significantly attenuated silica-induced cell death and IL-1ß release. PAX caused an increase in lysosomal pH and decrease in lysosomal proteolytic activity. PAX also caused a significant accumulation of lysosomal cholesterol. BK channel activators NS11021 and NS1619 increased silica-induced cell death and IL-1ß release. BK channel activation also caused a decrease in lysosomal pH and increase in lysosomal proteolytic function as well as a decrease in cholesterol accumulation. CONCLUSION: Taken together, these results demonstrate that inhibiting lysosomal BK channel activity with PAX effectively reduced silica-induced cell death and IL-1ß release. Blocking cytosolic K+ entry into the lysosome prevented LMP through the decrease of lysosomal acidification and proteolytic function and increase in lysosomal cholesterol.

A mouse model of wildfire smoke-induced health effects: sex differences in acute and sustained effects of inhalation exposures.

Due to climate change, wildfires have increased in intensity and duration. While wildfires threaten lives directly, the smoke has more far-reaching adverse health impacts. During an extreme 2017 wildfire event, residents of Seeley Lake, Montana were exposed to unusually high levels of wood smoke (WS) causing sustained effects on lung function (decreased FEV1/FVC). Objective: The present study utilized an animal model of WS exposure to research cellular and molecular mechanisms of the resulting health effects. Methods: Mice were exposed to inhaled WS utilizing locally harvested wood to recapitulate community exposures. WS was generated at a rate resulting in a 5 mg/m3 PM2.5 exposure for five days. Results: This exposure resulted in a similar 0.28 mg/m2 particle deposition (lung surface area) in mice that was calculated for human exposure. As with the community observations, there was a significant effect on lung function, increased resistance, and decreased compliance, that was more pronounced in males at an extended (2 months) timepoint and males were more affected than females: ex vivo assays illustrated changes to alveolar macrophage functions (increased TNFα secretion and decreased efferocytosis). Female mice had significantly elevated IL-33 levels in lungs, however, pretreatment of male mice with IL-33 resulted in an abrogation of the observed WS effects, suggesting a dose-dependent role of IL-33. Additionally, there were greater immunotoxic effects in male mice. Discussion: These findings replicated the outcomes in humans and suggest that IL-33 is involved in a mechanism of the adverse effects of WS exposures that inform on potential sex differences.

Self-replicating murine ex vivo cultured alveolar macrophages as a model for toxicological studies of particle-induced inflammation.

Alveolar macrophages (AM) are integral to maintaining homeostasis within the lungs following exposure to inhaled particles. However, due to the high animal number requirements for in vitro research with primary AM, there remains a need for validated cell models that replicate alveolar macrophages in form and function to better understand the mechanisms that contribute to particle-induced inflammation and disease. A novel, easily adaptable, culture model that facilitates the continued expansion of murine alveolar macrophages for several months, termed murine ex vivo cultured AM (mexAM) has been recently described. Therefore, the present work evaluated the use of mexAMs as a suitable model for primary AM interactions with nano- and micro-sized particles. mexAM displayed a comparable profile of functional phenotype gene expression as primary AM and similar particle uptake capabilities. The NLRP3 inflammasome-driven IL-1ß inflammatory response to crystalline silica and various nanoparticles was also assessed, as well as the effects of cationic amphiphilic drugs to block particle-induced inflammation. For all endpoints, mexAM showed a comparable response to primary AM. Altogether, the present work supports the use of mexAM as a validated replacement for primary AM cultures thereby reducing animal numbers and serving as an effective model for mechanistic investigation of inflammatory pathways in particle-induced respiratory disease.

Therapeutic treatment of dietary docosahexaenoic acid for particle-induced pulmonary inflammation in Balb/c mice.

OBJECTIVE AND DESIGN: The omega-3 polyunsaturated fatty acid docosahexaenoic acid (DHA) has been reported to suppress inflammation. Pulmonary inflammation can be directly linked to exposure of various occupational and man-made particles leading to pulmonary diseases. Therapeutic treatments are lacking for particle-induced pulmonary inflammation. These studies evaluated DHA as a therapeutic treatment for semi-acute and chronic particle-induced pulmonary inflammation. METHODS: Balb/c mice were oropharyngeal instilled with hydrophobic multi-walled carbon nanotube (MWCNT) or hydrophilic crystalline silica (SiO2) either as one instillation (semi-acute) or once a week for 4 weeks (chronic). One week later, the mice were placed on either a control or 1% DHA-containing diet for 3 weeks (semi-acute) or 12 weeks (chronic). Mice were assessed for inflammatory signaling within the lung lavage fluid, impact on phagolysosomal membrane permeability, shifts of macrophage phenotype gene expression (M1, M2a, M2b, and M2c), and pulmonary histopathology. RESULTS: DHA increased pulmonary inflammatory markers and lung pathology when mice were exposed to SiO2. There were trending decreases of inflammatory markers for MWCNT-exposed mice with DHA treatment, however, mostly not statistically significant. CONCLUSION: The anti-inflammatory benefits of DHA treatment depend upon the type of inflammatory particle, magnitude of inflammation, and duration of treatment.

Respiratory and systemic impacts following MWCNT inhalation in B6C3F1/N mice.

BACKGROUND: A very pure multi-walled carbon nanotube (MWCNT) that was shown to have very low toxicity in vitro, was evaluated for lung and systemic effects and distribution following inhalation exposure. METHODS: B6C3F1/N mice were exposed to varying doses (0, 0.06, 0.2, and 0.6 mg/m3) of the (99.1% carbon) MWCNT by inhalation for 30 days (excluding weekends). Ten days following the last exposure, the lungs and spleen were harvested and processed for histology and immune cell population assessment. In addition, lung lavage cells and fluid were analyzed. Stimulated Raman scattering (SRS) was used to identify particles in the lungs, spleen, kidneys, liver, mediastinal and brachial lymph nodes, and olfactory bulb. Splenic tissue sections were stained with hematoxylin and eosin (H&E) for light microscopic histopathology assessment. Blood plasma was analyzed for cytokines and cathepsins. A section of the spleen was processed for RNA isolation and relative gene expression for 84 inflammation-related cytokines/chemokines. RESULTS: Following MWCNT exposure, particles were clearly evident in the lungs, spleens, lymph nodes and olfactory bulbs, (but not livers or kidneys) of exposed mice in a dose-dependent manner. Examination of the lavaged lung cells was unremarkable with no significant inflammation indicated at all particle doses. In contrast, histological examination of the spleen indicated the presence of apoptotic bodies within T cells regions of the white pulp area. Isolated splenic leukocytes had significant changes in various cells including an increased number of proinflammatory CD11b+Ly6C+ splenic cells. The gene expression studies confirmed this observation as several inflammation-related genes were upregulated particularly in the high dose exposure (0.6 mg/m3). Blood plasma evaluations showed a systemic down-regulation of inflammatory cytokines and a dose-dependent up-regulation of lysosomal cathepsins. CONCLUSIONS: The findings in the lungs were consistent with our hypothesis that this MWCNT exposure would result in minimal lung inflammation and injury. However, the low toxicity of the MWCNT to lung macrophages may have contributed to enhanced migration of the MWCNT to the spleen through the lymph nodes, resulting in splenic toxicity and systemic changes in inflammatory mediators.

Docosahexaenoic acid impacts macrophage phenotype subsets and phagolysosomal membrane permeability with particle exposure.

Inhalation of particles results in pulmonary inflammation; however, treatments are currently lacking. Docosahexaenoic acid (DHA) is an omega-3 polyunsaturated fatty acid shown to exhibit anti-inflammatory capabilities. The impact of DHA on particle-induced inflammation is unclear; therefore, the aim of this study was to examine the hypothesis that DHA downregulates macrophage inflammatory responses by altering phagolysosomal membrane permeability (LMP) and shifting macrophage phenotype. Isolated Balb/c alveolar macrophages (AM) were polarized into M1, M2a, M2b, or M2c phenotypes in vitro, treated with DHA, and exposed to a multi-walled carbon nanotube (MWNCT) or crystalline silica (SiO2). Results showed minimal cytotoxicity, robust effects for silica particle uptake, and LMP differences between phenotypes. Docosahexaenoic acid prevented these effects to the greatest extent in M2c phenotype. To determine if DHA affected inflammation similarly in vivo, Balb/c mice were placed on a control or 1% DHA diet for 3 weeks, instilled with the same particles, and assessed 24 hr following instillation. Data demonstrated that in contrast to in vitro findings, DHA increased pulmonary inflammation and LMP. These results suggest that pulmonary responses in vivo may not necessarily be predicted from single-cell responses in vitro.

The role of lysosomal ion channels in lysosome dysfunction.

Lysosomes offer a unique arrangement of degradative, exocytic, and signaling capabilities that make their continued function critical to cellular homeostasis. Lysosomes owe their function to the activity of lysosomal ion channels and transporters, which maintain concentration gradients of H+, K+, Ca2+, Na+, and Cl- across the lysosomal membrane. This review examines the contributions of lysosomal ion channels to lysosome function, showing how ion channel function is integral to degradation and autophagy, maintaining lysosomal membrane potential, controlling Ca2+ signaling, and facilitating exocytosis. Evidence of lysosome dysfunction in a variety of disease pathologies creates a need to understand how lysosomal ion channels contribute to lysosome dysfunction. For example, the loss of function of the TRPML1 Ca2+ lysosome channel in multiple lysosome storage diseases leads to lysosome dysfunction and disease pathogenesis while neurodegenerative diseases are marked by lysosome dysfunction caused by changes in ion channel activity through the TRPML1, TPC, and TMEM175 ion channels. Autoimmune disease is marked by dysregulated autophagy, which is dependent on the function of multiple lysosomal ion channels. Understanding the role of lysosomal ion channel activity in lysosome membrane permeability and NLRP3 inflammasome activation could provide valuable mechanistic insight into NLRP3 inflammasome-mediated diseases. Finally, this review seeks to show that understanding the role of lysosomal ion channels in lysosome dysfunction could give mechanistic insight into the efficacy of certain drug classes, specifically those that target the lysosome, such as cationic amphiphilic drugs.

A contemporary review of electronic waste through the lens of inhalation toxicology.

Inhalation is a significant route of exposure to toxic chemicals for electronic waste (e-waste) workers, especially for those whose activities take place in the informal sector. However, there remains a dearth of research on the health effects produced by the hazardous dismantling of e-waste and associated outcomes and biological mechanisms that occur as a result of inhalation exposure. This contemporary review highlights a number of the toxicological and epidemiological studies published on this topic to bring to light the many knowledge gaps that require further research, including in vitro and ex vivo investigations to address the health outcomes and underlying mechanisms of inhaled e-waste-associated pulmonary disease.

Contribution of Particle-Induced Lysosomal Membrane Hyperpolarization to Lysosomal Membrane Permeabilization.

Lysosomal membrane permeabilization (LMP) has been proposed to precede nanoparticle-induced macrophage injury and NLRP3 inflammasome activation; however, the underlying mechanism(s) of LMP is unknown. We propose that nanoparticle-induced lysosomal hyperpolarization triggers LMP. In this study, a rapid non-invasive method was used to measure changes in lysosomal membrane potential of murine alveolar macrophages (AM) in response to a series of nanoparticles (ZnO, TiO2, and CeO2). Crystalline SiO2 (micron-sized) was used as a positive control. Changes in cytosolic potassium were measured using Asante potassium green 2. The results demonstrated that ZnO or SiO2 hyperpolarized the lysosomal membrane and decreased cytosolic potassium, suggesting increased lysosome permeability to potassium. Time-course experiments revealed that lysosomal hyperpolarization was an early event leading to LMP, NLRP3 activation, and cell death. In contrast, TiO2- or valinomycin-treated AM did not cause LMP unless high doses led to lysosomal hyperpolarization. Neither lysosomal hyperpolarization nor LMP was observed in CeO2-treated AM. These results suggested that a threshold of lysosomal membrane potential must be exceeded to cause LMP. Furthermore, inhibition of lysosomal hyperpolarization with Bafilomycin A1 blocked LMP and NLRP3 activation, suggesting a causal relation between lysosomal hyperpolarization and LMP.

Determination of the relative contribution of the non-dissolved fraction of ZnO NP on membrane permeability and cytotoxicity.

Background: While the role of lysosomal membrane permeabilization (LMP) in NP-induced inflammatory responses has been recognized, the underlying mechanism of LMP is still unclear. The assumption has been that zinc oxide (ZnO)-induced LMP is due to Zn2+; however, little is known about the role of ZnO nanoparticles (NP) in toxicity.Methods: We examined the contribution of intact ZnO NP on membrane permeability using red blood cells (RBC) and undifferentiated THP-1 cells as models of particle-membrane interactions to simulate ZnO NP-lysosomal membrane interaction. The integrity of plasma membranes was evaluated by transmission electron microscopy (TEM) and confocal microscopy. ZnO NP dissolution was determined using ZnAF-2F, Zn2+ specific probe. The stability of ZnO NP inside the phagolysosomes of phagocytic cells, differentiated THP-1, alveolar macrophages, and bone marrow-derived macrophages, was determined.Results: ZnO NP caused significant hemolysis and cytotoxicity under conditions of negligible dissolution. Fully ionized Zn2SO4 caused slight hemolysis, while partially ionized ZnO induced significant hemolysis. Confocal microscopy and TEM images did not reveal membrane disruption in RBC and THP-1 cells, respectively. ZnO NP remained intact inside the phagolysosomes after a 4 h incubation with phagocytic cells.Conclusions: These studies demonstrate the ability of intact ZnO NP to induce membrane permeability and cytotoxicity without the contribution of dissolved Zn2+, suggesting that ZnO NP toxicity does not necessarily depend upon Zn2+. The stability of ZnO NP inside the phagolysosomes suggests that LMP is the result of the toxic effect of intact ZnO NP on phagolysosomal membranes.

Mouse pulmonary dose- and time course-responses induced by exposure to nitrogen-doped multi-walled carbon nanotubes.

Objective: In this study, we compared in vitro and in vivo bioactivity of nitrogen-doped multi-walled carbon nanotubes (NDMWCNT) to MWCNT to test the hypothesis that nitrogen doping would alter bioactivity.Materials and Methods: High-resolution transmission electron microscopy (TEM) confirmed the multilayer structure of MWCNT with an average layer distance of 0.36 nm, which was not altered by nitrogen doping: the nanomaterials had similar widths and lengths. In vitro studies with THP-1 cells and alveolar macrophages from C57BL/6 mice demonstrated that NDMWCNT were less cytotoxic and stimulated less IL-1ß release compared to MWCNT. For in vivo studies, male C57BL/6J mice received a single dose of dispersion medium (DM), 2.5, 10 or 40 µg/mouse of NDMWCNT, or 40 µg/mouse of MWCNT by oropharyngeal aspiration. Animals were euthanized between 1 and 7 days post-exposure for whole lung lavage (WLL) studies.Results and Discussion: NDMWCNT caused time- and dose-dependent pulmonary inflammation. However, it was less than that caused by MWCNT. Activation of the NLRP3 inflammasome was assessed in particle-exposed mice by determining cytokine production in WLL fluid at 1 day post-exposure. Compared to DM-exposed mice, IL-1ß and IL-18 were significantly increased in MWCNT- and NDMWCNT-exposed mice, but the increase caused by NDMWCNT was less than MWCNT. At 56 days post-exposure, histopathology determined lung fibrosis in MWCNT-exposed mice was greater than NDMWCNT-exposed mice.Conclusions: These data indicate nitrogen doping of MWCNT decreases their bioactivity, as reflected with lower in vitro and in vivo toxicity inflammation and lung disease. The lower activation of the NLRP3 inflammasome may be responsible. Abbreviations: NDMWCNT: nitrogen-doped multi-walled carbon nanotubes; MWCNT: multi-walled carbon nanotubes; TEM: transmission electron microscopy; HRTEM: high resolution transmission electron microscopy; IL-1ß: interleukin-1ß; DM: dispersion medium; WLL: whole lung lavage; IL-18: interleukin-18; GSD: geometric standard deviation; XPS: X-ray photoelectron spectroscopy; SEM: standard error of the mean; PMA: phorbol 12-myristate 13-acetate; LPS: lipopolysacharride; LDH: lactate dehydrogenase; AM: alveolar macrophage; PMN: polymorphonuclear leukocyte.

Multiwalled Carbon Nanotubes of Varying Size Lead to DNA Methylation Changes That Correspond to Lung Inflammation and Injury in a Mouse Model.

Diversity in physicochemical properties of engineered multiwalled carbon nanotubes (MWCNTs) increases the complexity involved in interpreting toxicity studies of these materials. Studies indicate that epigenetic changes could be at least partially involved in MWCNTs-induced pro-inflammatory and fibrotic lung pathology. Therefore, we examined distinct methylation changes in response to MWCNTs of varied sizes to identify potential epigenetic biomarkers of MWCNTs exposure and disease progression. C57BL/6 mice were exposed via oropharyngeal instillation to a single dose (50 µg) to one of three differently sized MWCNTs: "narrow short" (NS), "wide short" (WS), and "narrow long" (NL). Vehicle-treated control mice received dispersion media (DM) only. Whole lung lavage fluid (LLF) and lung tissue were collected 24 h and 7 days postexposure to evaluate pro-inflammatory cytokines, epigenetic, or histological responses at acute and subchronic intervals, respectively. Luminometric methylation assay and pyrosequencing were used to measure global DNA methylation as well as promoter methylation of inflammation and fibrosis-related genes, respectively. Pro-inflammatory cytokines, including IL-1ß, IL-6, and TNF-α, were measured using enzyme-linked immunosorbant assay, while airway thickening and interstitial collagen accumulation were measured in 7-day lung tissue using laser scanning cytometry. Distinct patterns of methylation (i.e., IL-1ß, IL-6, and TNF-α) among the different sized MWCNTs at 24 h postexposure corresponded to some pro-inflammatory cytokine measurements from whole LLF. Fibrosis-related gene, Thy-1, was significantly hypermethylated after exposures to WS and NL MWCNTs, while only NL MWCNTs induced significantly lower global DNA methylation. After 7 days, a hierarchy in airway thickness and interstitial collagen deposition was observed: NS < WS < NL. However, only airway thickness was significantly greater in the WS and NL MWCNTs-exposed groups than the DM-exposed group. These data suggest that methylation changes could be involved in the initial immune response of inflammation and tissue remodeling that precedes lung disease in response to different MWCNTs sizes.

Using Time-Resolved Fluorescence Anisotropy of di-4-ANEPPDHQ and F2N12S to Analyze Lipid Packing Dynamics in Model Systems.

The fluorescence probes di-4-ANEPPDHQ and F2N12S have solvochromatic emission spectra and fluorescence lifetimes that are sensitive to order within the environment of lipid membranes. We show in this communication that the time-resolved fluorescence anisotropy of these probes, analyzed either by the wobble-in-a-cone model or by the model-independent order parameter S2, provides complementary information about dynamics and lipid packing in a variety of homogeneous lipid membranes systems.

Sex differences in the inflammatory immune response to multi-walled carbon nanotubes and crystalline silica.

Background: Respiratory disease is a leading cause of death and disability worldwide. These diseases frequently present with a sex bias in occurrence and severity, yet the mechanisms responsible for these sex biases is a critically understudied area of basic research. Methods: Male and female C57BL/6 mice were exposed to multi-walled carbon nanotubes (MWCNTs) or crystalline silica (cSiO2) via oropharyngeal aspiration. Acute assessments were conducted 24 h and 7 days after a single exposure. In chronic experiments, mice were exposed to respective particles once per week for 4 weeks and sacrificed 8 weeks after the last exposure. Lung lavage fluid (LLF) was assessed for markers of injury and inflammation. Immune cell populations were analyzed by flow cytometry and histopathology assessment was performed on lung tissue from chronically exposed mice. Results: Female mice exposed to a single dose of MWCNTs generated a greater eosinophilic response than males 24 h and 7 days post-exposure. Eosinophilia was accompanied by elevated type 2 cytokine production in LLF. The exaggerated acute response in females was consistent with lung pathology observed in the chronic model: females had greater alveolitis and epithelial cell hyperplasia compared to males. There were no sex differences 24 h after cSiO2 exposure, but by 7-day post-exposure female mice had greater airspace neutrophilia and inflammatory cytokine levels compared to males. However, following repeated exposure to cSiO2, male mice had worse alveolitis and greater dendritic cell presence within the lungs. Conclusions: Female mice are more susceptible to acute and chronic MWCNT-induced inflammation, but male mice are more susceptible to chronic cSiO2-induced lung pathology.

Prevention of crystalline silica-induced inflammation by the anti-malarial hydroxychloroquine.

Objectives: Inhalation of crystalline silica (cSiO2) remains a significant occupational hazard and may lead to the development of silicosis. When cSiO2 particles are phagocytized by alveolar macrophages, they cause disruption of the lysosomal membrane which results in cell death. There are currently no pharmaceutical treatments directed at this mechanism of disease; however, many existing pharmaceuticals, such as hydroxychloroquine (HCQ), become sequestered in the lysosome through an ion-trapping mechanism. The objective of this research was to determine whether HCQ can prevent cSiO2-induced toxicity by blocking LMP in alveolar macrophages. Materials and methods: This study assessed the ability of in vitro treatment with HCQ to block toxicity and lysosomal membrane permeability in cSiO2-exposed mouse bone-marrow derived macrophages. Additionally, C57Bl/6 mice were treated with HCQ by oral gavage before cSiO2 exposure, and the ability of HCQ to prevent lung injury and inflammation was assessed. Results:In vitro studies demonstrated that HCQ attenuated activation of the NLRP3 inflammasome and blocked LMP. Mice treated with HCQ in vivo showed a modest trend towards decreased cSiO2-induced toxicity. Ex vivo culture of alveolar macrophages collected from cSiO2-treated mice showed significantly less NLRP3 inflammasome activation after in vivo exposure to HCQ. Conclusions: Our findings suggest that hydroxychloroquine blocks LMP and can significantly decrease cSiO2-induced toxicity in vitro. HCQ may be a promising treatment for prevention of cSiO2-induced lung damage.

Length, but Not Reactive Edges, of Cup-stack MWCNT Is Responsible for Toxicity and Acute Lung Inflammation.

Multiwalled carbon nanotube (MWCNT) toxicity after inhalation has been associated with size, aspect ratio, rigidity, surface modification, and reactive oxygen species production. In this study, we investigated a series of cup-stacked MWCNT prepared as variants of the Creos 24PS. Mechanical chopping produced a short version (AR10) and graphitization to remove active reaction sites by extreme heat (2,800°C; Creos 24HT) to test the contribution of length and alteration of potential reaction sites to toxicity. The 3 MWCNT variants were tested in vitro in a human macrophage-like cell model and with C57BL/6 alveolar macrophages for dose-dependent toxicity and NLRP3 inflammasome activation. The 24PS and 24HT variants showed significant dose-dependent toxicity and inflammasome activation. In contrast, the AR10 variant showed no toxicity or bioactivity at any concentration tested. The in vivo results reflected those observed in vitro, with the 24PS and 24HT variants resulting in acute inflammation, including elevated polymorphonuclear counts, Interleukin (IL)-18, cathepsin B, and lactate dehydrogenase in isolated lung lavage fluid from mice exposed to 40 µg MWCNT. Taken together, these data indicate that length, but not the absence of proposed reaction sites, on the MWCNT influences particle bioactivity.

Modification of nano-silver bioactivity by adsorption on carbon nanotubes and graphene oxide.

OBJECTIVE: The toxicity of silver nanomaterials in various forms has been extensively evaluated, but the toxicity of silver nanocarbon composites is less well understood. Therefore, silver-carbon nanotube composites (Ag-MWCNT-COOH) and silver-graphene oxide composites (Ag-GO) were synthesized by microwave irradiation and evaluated in two in vitro cell models. MATERIALS/METHODS: Toxicity of silver nanosphere (Ag), Ag-MWCNT-COOH and Ag-GO were analyzed by MTS assay and LDH assay in primary C57BL/6 murine alveolar macrophages and human THP-1 cells. Activation of NLRP3 inflammasome by particle variants in these models was done by proxy using LPS co-culture and IL-1ß release. RESULTS: The results depended on the model, as the amount of Ag on the modified carbon resulted in slightly increased toxicity for the murine cells, but did not appear to affect toxicity in the human cell model. IL-1ß release from carbon particle-exposures was decreased by the presence of Ag in both cell models. Suspensions of Ag-MWCNT-COOH, Ag-GO and Ag in artificial lysosomal fluid were prepared and ICP-MS was used to detect Ag ions concentration in three silver suspension/solutions. The amount of Ag ions released from Ag-MWCNT-COOH and Ag-GO were similar, which were both lower than that of Ag nanospheres. CONCLUSIONS: The results suggest the bioactivity of silver composites may be related to the amount of Ag ions released, which can be dependent on the cell model under investigation.

The Effects of Varying Degree of MWCNT Carboxylation on Bioactivity in Various In Vivo and In Vitro Exposure Models.

Functionalization has been shown to alter toxicity of multi-walled carbon nanotube (MWCNT) in several studies. This study varied the degree of functionalization (viz., amount of MWCNT surface carboxylation) to define the relationship between the extent of carboxylation and effects in a variety of in vitro cell models and short-term ex vivo/in vivo particle exposures. Studies with vitamin D3 plus phorbol ester transformed THP-1 macrophages demonstrated that functionalization, regardless of amount, corresponded with profoundly decreased NLRP3 inflammasome activation. However, all MWCNT variants were slightly toxic in this model. Alternatively, studies with A549 epithelial cells showed some varied effects. For example, IL-33 and TNF-α release were related to varying amounts of functionalization. For in vivo particle exposures, autophagy of alveolar macrophages, measured using green fluorescent protein (GFP)- fused-LC3 transgenic mice, increased for all MWCNT tested three days after exposure, but, by Day 7, autophagy was clearly dependent on the amount of carboxylation. The instilled source MWCNT continued to produce cellular injury in alveolar macrophages over seven days. In contrast, the more functionalized MWCNT initially showed similar effects, but reduced over time. Dark-field imaging showed the more functionalized MWCNTs were distributed more uniformly throughout the lung and not isolated to macrophages. Taken together, the results indicated that in vitro and in vivo bioactivity of MWCNT decreased with increased carboxylation. Functionalization by carboxylation eliminated the bioactive potential of the MWCNT in the exposure models tested. The observation that maximally functionalized MWCNT distribute more freely throughout the lung with the absence of cellular damage, and extended deposition, may establish a practical use for these particles as a safer alternative for unmodified MWCNT.